SUBMISSION GUIDELINES

PLEASE NOTE NEW VOLUME AND CONCENTRATION REQUIREMENTS

Samples can be submitted in PCR strip tubes or PCR plates for Sanger sequencing using the following process and guidelines.  Your order request must be submitted online using dnaLIMS prior to physically submitting your samples:

Process

1

Prepare samples specific for the preferred service level, and follow the recommended protocol

SBC-Prepared
User-Prepared

2

Enter your order request online using dnaLIMS, and submit samples with a printed copy of your request form

 

3

Drop off in-person, by campus mail, or by courier to:

Sequencing and Bioinformatics Consortium
University of British Columbia
Room 3124 - PharmSci Building
2405 Wesbrook Mall
Vancouver, BC V6T 1Z3

Storefront Hours: 9am-9:30am
Monday, Tuesday, and Thursday
Meet at the Agronomy Road entrance to the Pharmaceutical Sciences building for non-contact sample drop off.   

 

Following these guidelines will ensure that your samples are processed without delay.

REMEMBER: Templates and primers should be resuspended in water or Tris (not TE), as EDTA will interfere with the ion concentration in the sequencing reaction. 

Process Service Levels: A-D

A: SBC-Prepared
  • Template and primer are submitted separately. 
  • Template:
    • We require a minimum of 10 µl of DNA sample at the concentration specified in the table below. 
    • If multiple reactions use the same template, please submit these as separate reactions (i.e. one 10 µl aliquot per reaction).
    • For orders with fewer than 48 samples, submit your samples in 8-strip PCR tubes. ONLY label the side of the tubes with your initials and sample number. For orders with 48 or more samples, submit your samples in a 96-well plate, filling in columns (e.g. A1 to H1). We can provide you with strips or plates if needed.
    • It is critical that the concentrations are measured carefully, as incorrect quantitation (whether higher or lower) will result in poor sequencing results. 

  • Primer:
    • If submitting custom primers, a minimum of 5 µl of primer at 5 pmol/µl is required per reaction.
    • Primers should be submitted in 1.5 ml eppendorfs, with the primer name labelled on the lid with indelible ink.
  • Templates and primers should be resuspended in water or Tris (not TE), as EDTA will interfere with the ion concentration in the sequencing reaction.
  • Recommended concentrations are listed in the table below.
Standard Templates Concentration Volume
plasmid DNA  40 - 250 ng/µl >10 µl per reaction
purified PCR product (100 - 300 bp) 2 - 20 ng/µl >10 µl per reaction
purified PCR product (301 - 1,000 bp) 3 - 40 ng/µl >10 µl per reaction
purified PCR product (over 1,000 bp) 20 - 200 ng/µl >10 µl per reaction
     
Other Templates    
cosmid/fosmid/BAC (over 20 kbp) 500 - 800 ng/µl >10 µl per reaction
     
Custom primer, if required*    
primer, submitted separately from template 5 pmol/µl >5 µl per reaction
     

*only required if a standard sequencing primer is not specified

Please read important information on template/primer considerations in our submission guidelines.

B: User-Prepared, Pre-Mixed
  • Template and primer are submitted pre-mixed with water. Our staff add sequencing chemistry, perform cycling, post-reaction clean-up, and electrophoreses.
  • We require 14 µl of sample, containing template and primer at the amounts specified in the table below, mixed with water as needed.
  • It is critical that the concentrations are measured carefully, as incorrect quantitation (whether higher or lower) will result in poor sequencing results.
Standard Template Final Amount Required Total volume
Plasmid DNA 400 ng 14 µl 
purified PCR product (100 - 300 bp) 10 ng 14 µl 
purified PCR product (301 - 1,000 bp) 30 ng 14 µl 
purified PCR product (over 1,000 bp) 100 ng 14 µl 
primer 10 pmol included in above
     
Other Templates    
cosmid/fosmid/BAC (over 20 kbp) 1200 ng 14 µl 
primer 10 pmol included in above
     

 

  • For orders with fewer than 48 samples, submit your samples in 8-strip PCR tubes. ONLY label the side of the tubes with your initials and sample number. For orders with 48 or more samples, submit your samples in a 96-well plate, filling in columns (e.g. A1 to H1). We can provide you with strips or plates if needed.
  • Templates and primers should be resuspended in water or Tris (not TE), as EDTA will interfere with the ion concentration in the sequencing reaction.
C: User-Prepared, SBC-Cleaned
  • User performs the cycle sequencing, and submits samples for post-reaction cleaning, injection, and electrophoretic separation.
  • We require 12 µl of cycled material. For orders with fewer than 48 samples, submit your samples in 8-strip PCR tubes. Label the side of the tubes with your initials and sample number. For orders with 48 or more samples, submit your samples in a 96-well plate, filling in columns (e.g. A1 to H1). We can provide you with strips or plates if needed.
  • Templates and primers should be resuspended in water or Tris (not TE), as EDTA will interfere with the ion concentration in the sequencing reaction.
D: User-Prepared, User-Cleaned
  • User performs the cycle sequencing and post-reaction clean-up, and submits samples for injection and electrophoretic separation.
  • We require 12 µl of sample resuspended in water, not formamide. For orders with fewer than 48 samples, submit your samples in 8-strip PCR tubes. Label the side of the tubes with your initials and sample number. For orders with 48 or more samples, submit your samples in a 96-well plate, filling in columns (e.g. A1 to H1). We can provide you with strips or plates if needed.
  • Templates and primers should be resuspended in water or Tris (not TE), as EDTA will interfere with the ion concentration in the sequencing reaction.

 

Protocol: Standard Sequencing (Plasmid, PCR Product)
Reaction Set-up

Plasmid DNA 

Template  200 ng
Primer 5 pmol
BigDye® dilution mix 3  µl
dH2O to final volume: 10 µl

 

PCR Product (choose product length)

Template (100-300bp) 5 ng
OR (301-1000bp) 15 ng
OR (over 1000bp) 50 ng
Primer 5 pmol
BigDye® dilution mix 3 µl
dH2O to final volume: 10 µl

BigDye® dilution mix can be purchased from SBC (please see services and pricing)

Cycling Protocol

(ramp rate = 1°C/sec)

hot start 96°C 1 min
25x 96°C 10 sec
50°C 5 sec
60°C 4 min
soak 4°C 8

 
.

Protocol: Specialty Sequencing (COSMID, BAC)
Reaction Set-up

Cosmid/BAC

Template 600 ng
Primer 5 pmol
BigDye® dilution mix 3 µl
dH2O to final volume: 10 µl

BigDye® dilution mix can be purchased from SBC (please see services and pricing)

Cycling Protocol
hot start 96°C 1 min
50x 96°C 10 sec
50°C 5 sec
60°C 4 min
soak 4°C 8

 


 

Template and Primer Requirements

Template Considerations

Of primary importance to the success of DNA sequencing reactions is the quality and quantity of sample provided. SBC cannot be held responsible for failures in sequencing which are related to poor preparation of the DNA template used, or failure to follow recommended guidelines.

  • Contaminated template will give high background noise and poor, or no, sequence information
  • Many of the commercially-available extraction and purification kits (eg. Qiagen, Promega, Wizard Plus, Sigma) are recommended
  • *** please ensure that the elution buffer does NOT contain EDTA ***
  • It is critical that the concentrations are measured carefully, as incorrect quantitation (whether higher or lower) will result in poor sequencing results. 
  • Modified conditions available for “difficult” templates (GC-rich, poly-T)

SBC-Prepared Requirements |  User-Prepared Requirements

Primer Considerations

Recommendations when designing custom sequencing primers:

  • Tm between 55°C and 60°C
  • Formula: ºC = 4(G+C) + 2(A+T)
  • “G” or “C” at 3’end
  • avoid primers with runs of 3 or more of the same base
  • use primer design software to avoid primers with hairpin or dimer characteristics

Custom primers must be submitted at a concentration of 5pmol/µl, resuspended in Tris or dH2O (NOT EDTA)

Standard Sequencing Primers available at SBC

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